Development of Tobacco Varieties with No or Significantly Reduced Anatabine Content

ABSTRACT

A process for producing a reduced tobacco-specific nitrosamine (TSNA) tobacco plant comprising reducing and/or eliminating anatabine biosynthesis in wild-type tobacco plant. In addition, use of such plants for discovery of molecular markers that are closely linked with genes required for anatabine biosynthesis and for discovery of genes required for anatabine biosynthesis. In addition, a smoking composition, a smoking article and a smokeless tobacco oral delivery product contain the tobacco material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/529,307, filed Aug. 1, 2019, which is a Continuation of U.S. patentapplication Ser. No. 14/207,082, filed on Mar. 12, 2014 (now U.S. Pat.No. 10,375,910, issued Aug. 13, 2019), which claims the benefit of U.S.Provisional patent application Ser. No. 61/799,831, filed on Mar. 15,2013. All three applications are incorporated herein by reference intheir entireties for all purposes.

BACKGROUND

Nicotiana tabacum produces several different pyridine alkaloids, withnicotine representing the most abundant. The alkaloids that accumulateto a lesser degree include anatabine, nornicotine, myosmine, andanabasine. These alkaloids are commonly referred to as minor alkaloids.Minor alkaloids are direct precursors in the formation of tobaccospecific nitrosamines.

Nitrosamines and in particular, tobacco specific nitrosamines (TSNAs)are constituents of tobacco. Anatabine, a minor alkaloid, is nitrosatedto form N′-nitrosoanatabine (NAT). NAT constitutes approximately 40% oftotal TSNAs. The biosynthesis of anatabine, and its associated genes, isnot completely known. However, current understanding is that thesynthesis of anatabine requires quinolinate phosphoribosyltransferease(QPT), a key enzyme regulating entry to the pyridine nucleotide cycle.

Biosynthesis of anatabine is thought to proceed via the conversion ofnicotinic acid to 3,6-dihydronicotinic acid. After decarboxylation toproduce 3,6-dihydropyridine, and condensation with nicotinic acid toproduce an intermediate, conversion to anatabine can occur bydehydrogenation. The current understanding of the alkaloid biosyntheticpathway is depicted in FIG. 1 .

In addition to TSNAs, certain polyphenol compounds can form undesirablephenolic compounds during the combustion of tobacco and may also betargeted constituents of tobacco smoke. There is interest in providing amethod for reducing the contents of these targeted compounds in tobacco.

BRIEF SUMMARY

The present disclosure significantly reduces and/or eliminates anatabinebiosynthesis in tobacco. This prevents anatabine, a precursor ofN′-nitrosoanatabine (NAT), from accumulating. NAT representsapproximately 40% of total Tobacco Specific Nitrosamines (TSNAs), and asa result of elimination of anatabine as precursor, a 40% reduction intotal TSNAs will be possible. This “ultra-low anatabine” trait can becombined with an ultra-low nornicotine trait in a tobacco line orvariety to accomplish a further reduction of TSNA accumulation, asnornicotine is a precursor for N′-nitrosonornicotine formation. Thecombined ultra-low anatabine and ultra-low nornicotine traits couldresult in a reduction of total TSNAs on the order of about 70-80%.

The present disclosure details the development of an ultra-low anatabinetrait through induced variation (EMS treatment) followed by a screen forplants deficient in anatabine. The ultra-low anatabine trait can becrossed into other commercial varieties, or the trait can be induced ina similar fashion directly into commercial tobacco varieties.

Currently there is no commercially relevant tobacco variety with reducedanatabine in leaf lamina. Since anatabine is a precursor for NAT whichconstitutes—40% of total TSNAs, this trait has applicability inproducing tobacco with very low TSNAs for tobacco products.

The ultra-low anatabine plants are also ideal genetic material fordiscovery of molecular markers that are closely linked with genesrequired for anatabine biosynthesis, as well as the actual discovery ofgenes required for anatabine biosynthesis. As disclosed, a method fordiscovery of molecular markers that are closely linked with genesrequired for anatabine biosynthesis may comprise crossing a reduced TSNAtobacco plant with another tobacco plant, isolating genomic DNA fromindividual progeny of the cross, and detecting in the genomic DNA thepresence or absence of a molecular marker that is closely linked with agene required for anatabine biosynthesis. Also as disclosed, a methodfor discovery of genes required for anatabine biosynthesis may comprisecrossing a reduced TSNA tobacco plant with another tobacco plant,isolating genomic DNA from individual progeny of the cross, anddetecting in the genomic DNA the presence or absence of a gene requiredfor anatabine biosynthesis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the alkaloid biosynthetic pathway.

FIG. 2 shows the reduction of anatabine content compared to other mutantlines.

FIG. 3 illustrates the percent anatabine content present in selectedFC401 mutant lines at different generations.

FIG. 4 illustrates that the total anatabine content present in aselected TN90 LC mutant line at M2 and M3 generations.

FIG. 5 illustrates that the low anatabine FC401 M2 mutant line showsreduced NAT.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, “tobacco material” denotes a tobacco starting materialto be treated in various processes described herein, regardless of type,source or origin, which may have previously been subjected to othertreatments. The tobacco material may include, but is not limited to,tobacco solids and any solid form of tobacco, such as, e.g., curedtobacco (such as flue-cured tobacco); uncured tobacco (also known asgreen tobacco); dried, aged, cut, ground, stripped or shredded tobacco;tobacco scrap; expanded tobacco; fermented tobacco; reconstitutedtobacco; blended tobacco, etc. The tobacco material may be from anyparts of the tobacco plant, such as leaf, stem, veins, scrap and wastetobacco, cuttings, etc.

In one embodiment, the smoking article is a cigarette.

Preferably, smokeless tobacco oral delivery products, such as chewingtobacco or pouched tobacco, are sized to comfortably be received in ahuman mouth. In addition, the oral products may be sized so that it canbe moved around inside a human mouth.

A pouched tobacco typically contains an external wrapper and a tobaccomaterial therein. The external wrapper preferably comprises a membranethat is sufficiently porous to allow passage through the membrane of aliquid, such as saliva, in the mouth. The external wrapper membrane ispreferably resistant to deterioration in the presence of saliva andbacteria, and may be constructed from cellulose fiber such as tea bagmaterial.

One embodiment provides a method of producing a reduced tobacco-specificnitrosamine (TSNA) tobacco plant comprising reducing and/or eliminatinganatabine biosynthesis in wild-type tobacco plant. This may includepreventing the accumulation of anatabine, the precursor ofN′-nitrosoanatabine (NAT). This method may also comprise crossing saidtobacco plant with reduced and/or eliminated anatabine biosynthesis witha tobacco plant having low nornicotine synthesis. A further embodimentis a reduced TSNA tobacco plant developed from these methods or a plantcell line produced from these methods. The TSNA levels can be reduced byat least about 40% when compared to a wild-type tobacco plant. Furtherenvisioned is where the TSNA levels are reduced by at least about 70%when compared to a wild-type tobacco plant.

Another embodiment is the reduced TSNA tobacco plant in which a portionof said plant is used in a consumable tobacco product. This tobaccoproduct can be, e.g., a cigarette or a smokeless tobacco product madefrom a reduced TSNA tobacco plant. Another embodiment is seeds from thereduced TSNA tobacco plant comprising reduced levels of anatabine.

Another embodiment provides a method for discovery of molecular markersthat are closely linked with genes required for anatabine biosynthesisor discovery of genes required for anatabine biosynthesis comprisingcrossing a reduced tobacco-specific nitrosamine tobacco plant withanother plant, isolating genomic DNA from individual progeny of thecross, and detecting in the genomic DNA the presence or absence of amolecular marker that is closely linked with a gene required foranatabine biosynthesis or a gene required for anatabine biosynthesis.

The embodiments disclosed herein are further illustrated by thefollowing specific examples but is not limited hereto.

EXAMPLES Example I

A novel genetic variation in a population of tobacco plants was createdto identify plants that have lost the ability to biosynthesizeanatabine. These plants are very likely to have a mutated non-functionalversion of one or more genes critical for anatabine biosynthesis. Toinduce novel genetic variation plants were treated with Ethyl methanesulfonate (EMS) and propagated to the M2 stage so that recessivemutations would express in plants homozygous for mutated genes.

A population of the Flue-cured variety “401” and TN90 was available forthis investigation. Approximately 5000 seeds per variety were treatedwith 0.8% ethyl methane sulfonate and germinated. M1 plants were grownin the field and M2 seeds were collected. Approximately 2000 FC401 M2and 500 TN90 M2 seeds were germinated and grown in 6″ pots. At 50%flowering stage, plants were topped and leaf samples were collectedafter 2 weeks of topping.

Alkaloids were extracted from the leaf samples: 1 ml methanol per gramtissue was added and sonicated for 30 minutes. The extract wascentrifuged briefly to pellet the residual leaf tissue and purifiedextract was concentrated 10 times by vacuum centrifugation forapproximately 75 minutes at 45° C. Five microliters of concentratedsample along with standards were loaded on HPTLC plates. After sampleswere dried for 20 minutes, the plate was transferred quickly into thedeveloping chamber saturated with a solvent mixture of Toluene: Ethylacetate: Diethylamine (5:4:1 respectively). The plate was run forapproximately 10-15 minutes until the mobile phase ran throughapproximately 75% of the length of the plate. The plate was air driedand photographed under UV254 using a documentation system with video(CAMAG REPRO STAR 3). This image is reproduced in FIG. 2 .

Alkaloid Analysis: Tobacco leaves were harvested and air-dried in anoven at 50° C. A one gram sample of crushed, dried leaf was added to 10mL of 2 N NaOH in an extraction bottle. The sample was mixed and allowedto incubate for 15 minutes at room temperature. Alkaloids were extractedby the addition of 50 mL of extraction solution [0.04% quinolone(wt/vol) dissolved in methyl-tert-butyl ether] and gently rotated on alinear shaker for 3 hours. Following phase separation, an aliquot of theorganic phase was transferred to a sample vial. Samples were analyzedusing an Agilent 6890 Gas Chromatograph and 5973N Mass Spectrometer.

A total of 726 FC401 and 500 FN90 leaf samples were processed andscreened for anatabine mutants. Mutant plants 97 (08GH97) and 153(08GH153) in FIG. 2 show the reduction of anatabine content compared toother mutant lines. Using HPTLC screening and GCMS, 4 mutant lines wereidentified from FC401 and TN90 (Table 1). Mutants from dMS932 (ATCC®Accession Number PTA-124990), MF445 from FC401, and MS 3908 from TN90mutant seeds were used for further analysis.

TSNA Analysis: Tobacco leaves were harvested and freeze-dried andpowdered to 1 mm size. A 750 mg powdered sample was added to 30 mL of100 mM ammonium acetate in a 60 mL amber vial. The sample was mixed andincubated in a shaker for 30 minutes. Approximately 4 mL of sample wastransferred directly into labeled disposable culture tubes and 0.250 mLof concentrated ammonium hydroxide was added. The sample was vortexedfor 1-5 seconds, after which time 1.5 mL of sample was added to apreconditioned extraction cartridge with a flow rate of 1-2 drops persecond. Cartridges were washed and dried. Analytes from the extractioncartridges were eluted using 1.5 mL of 70:30 methanol:0.1% acetic acidand analyzed using liquid chromatography with tandem mass spectrometry(LC/MS/MS).

TABLE 1 Alkaloid Analysis by HPTLC and GC/MS Total Nicotine NornicotineAnabasine Anatabine Alkaloids % Anatabine Variety Seed ID Plant ID (%/gdry wt) (%/g dry wt) (%/g dry wt) (%/g dry wt) (%/g dry wt) (%/g dry wt)FC401 MS108 08GH97 1.46  0.0412 0.00429 0.0115 1.517 0.76 mutant 1(dMS932) FC401 MS445 08GH361 2.15  0.136  0.00423 0.0216 2.312 0.93mutant 2 FC 401 MS170 08GH153 1.190 0.0224 0.00420 0.0084 2.312 0.93mutant 3 FC 401 FC401 08GH1625 1.97  0.0476 0.0102  0.0875 2.115 4.41Control TN90 MS3908 09MN8938 4.08  0.092  0.0047  0.021  4.198 0.51mutant 1 TN90 TN90 LC 09N26 4.44  0.118  0.0129  0.123  4.694 2.62control Control

FIG. 3 shows consistently low anatabine levels in mutagenized lines ofthe FC401 tobacco strain through multiple generations. Note that thedata in M2 is from a single plant, while M3 and M4 are average valuesfrom multiple plants. FIG. 4 shows consistently low anatabine levels ina mutagenized line of the Tennessee Burley 90 (TN90) strain throughmultiple generations. As with FIG. 3 , the data in M2 is from a singleplant, while M3 is data of average values from multiple plants.

FIG. 5 shows the result of 750 M2 plants screened using High PerformanceThin Layer Chromatography (HPTLC). 12 plants were selected duringinitial screening and mutants containing the least anatabine arepresented. Control plants: Total alkaloid content in FC401 is 2-3%;percent anatabine is 2.5-3% and NAT ranges from 0.2-0.4 PPM. Anatabinemutant: Total alkaloid content 3.16%; percent anatabine 0.5% and NAT is0.051%. i.e˜over 80 percent reduction.

While the foregoing has been described in detail with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications may be made, and equivalentsthereof employed, without departing from the scope of the claims.

All of the above-mentioned references are herein incorporated byreference in their entirety to the same extent as if each individualreference was specifically and individually indicated to be incorporatedherein by reference in its entirety.

Deposit Information: A deposit of the proprietary Nicotiana tabacumdMS932 line disclosed above and recited in the appended claims has beenmade with American Type Culture Collection (ATCC®), 10801 UniversityBoulevard, Manassas, Va. 20110, USA. The date of deposit for N. tabacumline dMS932 was Feb. 21, 2018. The deposit of 100 packets eachcontaining 25 seeds (2500 total seeds) was taken from the same depositsmaintained since prior to the filing date of this application. Uponissuance of a patent, all restrictions upon the deposits will beirrevocably removed, and the deposits are intended by Applicant to meetall of the requirements of 37 C.F.R. §§ 1.801-1.809. ATCC® has issuedthe accession number: ATCC® Accession No. PTA-124990 for this deposit ofN. tabacum line dMS932. This deposit will be maintained in thedepository for a period of 30 years, or 5 years after the last request,or for the effective life of the patent, whichever is longer, and willbe replaced as necessary during that period. Applicant does not waiveany infringement of their rights granted under this patent or under thePlant Variety Protection Act (7 U.S.C. 2321 et seq.)

1.-20. (canceled)
 21. A pouched tobacco product comprising tobaccomaterial from a tobacco plant from tobacco plant line dMS932, wherein arepresentative sample of seed of said tobacco plant line is depositedunder ATCC® Accession No. PTA-124990.
 22. The pouched tobacco product ofclaim 21, wherein the pouched tobacco product is sized to be received ina human mouth.
 23. The pouched tobacco product of claim 21, wherein thepouched tobacco product is sized to be moved around inside a humanmouth.
 24. The pouched tobacco product of claim 21, wherein the pouchedtobacco product contains an external wrapper and the tobacco materialtherein.
 25. The pouched tobacco product of claim 24, wherein theexternal wrapper comprises a membrane.
 26. The pouched tobacco productof claim 25, wherein the membrane is sufficiently porous to allowpassage of a liquid through the membrane.
 27. The pouched tobaccoproduct of claim 26, wherein the liquid is saliva.
 28. The pouchedtobacco product of claim 25, wherein the membrane is resistant todeterioration in the presence of saliva.
 29. The pouched tobacco productof claim 25, wherein the membrane is resistant to deterioration in thepresence of bacteria.
 30. The pouched tobacco product of claim 25,wherein the membrane is constructed from cellulose fiber.
 31. Thepouched tobacco product of claim 21, wherein the tobacco material iscured tobacco material.
 32. The pouched tobacco product of claim 31,wherein the cured tobacco material is flue-cured tobacco material. 33.The pouched tobacco product of claim 21, wherein the tobacco material isselected from the group consisting of uncured tobacco material, driedtobacco material, and aged tobacco material.
 34. The pouched tobaccoproduct of claim 21, wherein the tobacco material is selected from thegroup consisting of ground tobacco material, stripped tobacco material,and shredded tobacco material.
 35. The pouched tobacco product of claim21, wherein the tobacco material comprises material from tobacco leaf.36. The pouched tobacco product of claim 21, wherein the tobaccomaterial comprises material from tobacco stem.